3,5DepartmentofBiotechnology,ShooliniUniversity,Solan-173230,India

Biologically
synthesized silver nanoparticles are extensively used in the field of
medicines, chemistry, zoology, botany, physics and other area of research.
AgNPs are well known to have inhibitory and bactericidal activity. In the
present research, biogenic green synthesis of silver nanoparticles (AgNPs)
using medicinally important plant Anacyclus pyrethrum extract has been
carried out. No additional toxic chemical was used in the process of reduction
and capping of nanoparticles. Synthesized AgNPswas characterized by UV-Vis
spectroscopy, X-rays diffraction, FTIR and Transmission electron microscopy.
TEM images represents that the synthesized nanoparticles showed spherical
geometry with an average size of 14 to 34 nm. FTIR studies shows the
responsible functional groups of biomolecules present in the plant extract for
reduction and capping of nanoparticles. The XRD spectra shows that the AgNPs
are of face centered cubic (FCC) structure. Silver nanoparticles synthesized by
Anacyclusphythrumalso have a great potential against bacteria P. aeruginosa,
followed by S. Typhi, and K. Pneumonia, respectively. So that
plant mediated synthesis of nanoparticles has been suggested as a cost
effective and environment friendly process and alternative to chemical and physical
methods.

Nano
range microscopic particles are the excellent research area of interest because they are bridging the gap
between the bulk material and molecular or atomic structure level.

By literature review, many physical and chemical methods
such as sol-gel process, chemical precipitation, reverse micelle method, etc. were
found for the synthesis of metallic nanoparticles [4].

Nowadays, biological and green methods are used for the production
of nanoparticles by using medicinal plants, fungi, and bacteria. Physiochemical
methods required lots of hazardous and toxic chemicals and are highly expensive
and time-consuming process [5]. The new green approach is developed to overcome
this kind of problems by the researchers. Nature has blessed us with an enormous
wealth of herbal plants which are widely distributed all over the world as a
source of therapeutic agent [6] and for the prevention and cure of various
diseases [7]. Therefore, in this study an attempt has been made to explore such
plants and form the Nanoparticlesto make a significant contribution to the
society. Plants seem to be the Nano factories for the production of
nanoparticles [8]. Plants extracts contain various types of secondary
metabolites having reducing as well as capping agent’s properties for the
production of nanoparticles. It is a very reliable and environment-friendly
process without using any toxic chemicals. Silver nanoparticles have wide uses
in different regions such as in nanobiotechnologicalresearch[9-10],
sensors[11], catalysis[12], cell electrodes[13], low-cost paper batteries [14]
etc. They are used as antimicrobial agents in wound dressings [15-17] as
topical creams to prevent wound infections [18], and as anticancer agents [19].

Anacyclus
pyrethrum (common name Akarkara) is a
medicinally crucialperennial herb that belongs to family Asteraceae
found in Indian subcontinent region, in the Himalayas, in northern India, North
in Arabian countries. It is mainly known for its aphrodisiac effect on the
body, and it also helps in the detoxification of excess waste and fluids from
the body. Chemical obtained from this plant are pyrethrin, Inulin, alkyl
amides, Anayclin, sesamin,andhydrocarolin. The alkyl amides are responsible for
its aphrodisiac and neuroprotective properties.

Anacyclus
pyrethrum root is used for atoothache,
rheumatic and neuralgic affections and rhinitis. Ayurveda Pharmacopoeia of
India indicates the use of the root in sciatica, paralysis, hemiplegic and
amenorrhea [20]. Further, in our study these biologically synthesized
nanoparticles were studied against different bacterial species to evaluate
their antimicrobial efficacy. So that the main aim of this study is the focused
on formulation of nanomaterials with the help of plant extracts which includes
the enhancement of solubility, protection from toxicity, enhancement of
pharmacology activity and protection from physical and chemical degradation.

2.
MATERIALS AND METHODS:

2.1
Collection of Plant material:

The
healthy plant materials of Anacyclus pyrethrum were collected from Patanjali
herbal garden, HaridwarUttarakhand. All the analytical reagents were purchased
from Fisher chemicals. Double distilled water was used throughout the
experiments.

2.2 Preparation of plant extract:

For
the green synthesis of silver nanoparticles, 10gm of roots were thoroughly
washed with running tap water, followed by distilled water and dried at room
temperature. Dried and powdered roots material was boiled with 100ml of
Millipore water at 60o for 30 min as reported earlier. The extract
was cooled to room temperature and filtered by Whatman filter paper No.1 and
finally stored at 4oC for further experiment.

Figure1.Shows theplantandrootsofAnacycluspyrethrum

2.3
Preparation of silver nanoparticles:

For
the synthesis of silver nanoparticles, 10 ml of aqueous roots extract was added to 90 ml of AgNO3 (2 mM)
solution and was kept for 48 hours for the formation of AgNPs. The color
changes of root extract indicated the formation of AgNPs. The reaction mixture was
centrifuged at 10,000 rpm for 15 minutes. A pellet was collected followed by
redispersion of pellets of AgNPs in deionized water to get rid of any
uncoordinated biological impurities.

2.4
UV-VIS spectra analysis:

UV-VIS
spectrum of synthesized AgNPs was carried out at different interval of time.
The bioreduction of Ag+ to Ag0 and stability using roots
extracts was monitored by periodic sampling of aliquots (1 ml) of aqueous
component. The AgNPs show the Plasmon resonance at 430 nm[22]. From the study,
it has been found that silver nanoparticles show the characteristic SPR at a
wavelength in the range of 400-450 nm. Broadening of peak indicated that the
particles are polydispersed[23].

2.5.
X-ray diffraction analysis of AgNPs:

The synthesis and crystalline
nature of nanoparticles were confirmed by X-ray diffraction (XRD) method. The
particles size of AgNPs was confirmed by using Debye Sherrer's equation.

D=0.94λ/βcosθ:

Where
D is the average crystalline domain size perpendicular to the reflecting
planes, λ is the wavelength of X-rays, β is the full width at half
maximum (FWHM), and θ is the diffraction angle.

2.6.TEManalysisofsynthesizedAgNPs:

Structural
Morphology and size of the synthesized silver nanoparticles were identified by
TEM images using PUSA TEM 91/N-III, JEOL TEM 1011 TEM 100 KVA instrument. A
thin film of the sample was prepared on a carbon coated copper grid by just
dropping a tiny amount of the sample on the grid and drying under thelamp.
Hence the size, shape and phase composition of particles were studied by TEM.

2.7.FTIR:

FT-IR
analysis was carried out by using PerkinElmer FT-IR C91158 Spectrum to identify
the bioactive compounds of Anacyclus pyrethrum roots extract to reduce
Ag+ ions and capping of the silver nanoparticles associated with synthesized
AgNPs and spectrum was recorded from 4000 cm-1 to 400 cm-1.

Evaluationofantimicrobialactivityof Nanoparticles:

Antimicrobial
activity of various nanoparticles was assessed by well diffusion method [24].
The turbidity of the subcultured microorganisms was adjusted with sterile distilled
water using 0.5 McFarland as standard (~1.5 X 108 cells/ml). Mueller
Hinton Agar (HiMedia) was prepared by dissolving readymade agar powder in
distilled water. Agar plates were prepared and inoculated with the test
microorganisms by spread plate method. The plates were left undisturbed for 30
min at room temperature. The powdered nanoparticles were weighed and dissolved
in Dimethyl Sulfoxide (DMSO) and used in triplicates. Then, the solution was
added in the wells in a constant concentration of 50µl/well. The standard of
antibiotic Chloramphenicol (HiMedia) was also prepared as a positive control.
DMSO was also added to a well as negative control to make sure that the solvent
used for dissolving the extracts do not have antimicrobial activity. Then, the
plates were incubated at 37°C for 24h in an upright position. After incubation,
the zone of inhibition was measured and compared with the standard antibiotic zone.

Minimum
Inhibitory Concentration:

The
MIC assay was performed for those nanoparticles which were active by well
diffusion susceptibility assay (inhibition zone >10 mm) [25, 26]. The MIC
values were determined by microdilution method. The plates were prepared by
dispensing 100μl of nutrient broth into each well. 100μl was taken
from the stock solution of tested nanoparticles (concentration of 50mg/ml) and
added into the first well of the plate. Then, two-fold serial dilutions were
performed by using a micropipette. The obtained concentration range was from 50
mg/ml, and then added 50μl of inoculum to each well except negative
control. The positive control of antibiotic (Chloramphenicol), negative control
(Nutrient broth), Broth alone and the inoculums alone were also put in the
experiment. The test plates were incubated at 37°C for 24h. The lowest sample
concentration showing clear well and inhibited complete growth were taken as
MIC value [27].

3. RESULT AND DISCUSSION:

3.1UV-Visiblespectroscopy:

UV-Visible
spectroscopy is one of the key tools to study the synthesis of metal
nanoparticles in aqueous solution. The reduction of silver metal ions inanaqueous
solution by using roots extract of Anacyclus pyrethrum was monitored by
the color change, i.e., colorless to reddish brown with the help of UV–Vis
spectroscopy.

The
characteristic SPR of colloidal Ag nanoparticles ranges between 300 to 500 nm.
(Fig. 1). UV-Vis absorption spectra (fig. 1) represented that the broad SPR
contained one peak at 420 nm. It is well known that AgNPs exhibit different colors
depending on the size of the AgNPs and these are due to the excitation of SPR
in the AgNPs. The SPR absorbance is sensitive to the plant extract
concentration nature, size and shape of particles present in the solution.

Figure 1.Uv-Visible peak of
AgNPs Solution at 420 nm

3.2
X-ray diffraction analysis of synthesized AgNPs:

Figure
2 shows the XRD diffraction pattern corresponding to reduced silver. Intense
peaks were observed at 2θ value is equal to 38.37, 44.60, 64.91, 77.99 and
could be recognized to the 111, 200, 220 and 311 crystallography planes of the face
centered cubic (fcc) silver crystal, respectively. The average crystalline size
is calculated using Debye Scherrer formula and the calculated average size of
silver nanoparticles is 14 nm.

Position [°2Theta] (Copper (Cu))

Figure
2. XRD peak pattern of synthesized silver nanoparticles

3.3. FTIR:

FTIR
spectrum of the roots extract shows peaks at about 3412, 2916, 2328, 1640,
1351, 1255 and 1039 cm-1. Peak at 3412 cm-1 arises due to
N-H stretching of anamino group or is indicative of O-H group due to the presence
of alcohols, phenols. Peak at 2916 cm-1indicates the presence of C-H
bond stretching of the alkyl group. Peaks at 1640 cm-1are associated
with N-H bond in amino acid. Peak at 1351 cm-1 represents C-N stretch vibration
as well as to amide I bands of proteins in the roots extract. The band at 1255
cm-1 confirms the presence of C–O groups from polyols. C-O stretch assigned to
alcohols represented by peak at 1039 cm-1

Figure 3.represents the
FTIR Analysis of silver nanoparticles

3.4.TEManalysisofsynthesizedAgNPs:

TEM
micrograph revealed that the particles are spherical, oval and well dispersed
without agglomeration (fig.3). The particles size of synthesizes silver nanoparticles
from Anacyclus pyrethrum roots extract is in the range of 19-34 nm.
Various reports have provided evidence of extracellular synthesis of silver nanoparticles
by TEM images.

3.5
Antibacterial property of synthesized AgNPs:

AgNPs
have lately received agreat deal of attention and concern due to their antibacterial activity. In the present study,
the biologically synthesized AgNPs from Anacyclus pyrethrum showed
excellent antimicrobial activity against test microorganisms. (table1. The
antibacterial activity of silver nanoparticles against the human pathogens
showed varied levels of inhibition. The zone of inhibition of AgNPs for P. aeruginosa,
K. pneumonea, and S. Typhi. were 11.2 ± 0.5; 10.0 ±1.2, 10.6 ±1.5
0.2 mm, respectively. As shown in Table-1, the present study revealed that
AgNPs possess potential antibacterial activity against, but it was
inactive against S. aureus.

The
results obtained by measurement of the zone of inhibition were presented in
table-1. As shown in table 1 and fig. 4, the nanoparticles showed a maximum
zone of inhibition against P. aeruginosa, followed by S. Typhi,
and K. Pneumoniae respectively.

Determination of MIC:

The
maximum inhibitory concentration of the
AgNPswas estimated by microdilution method was used. The lowestconcentration of
AgNPs at which there is no visible growth of the organism is mentioned in Table
2.

CONCLUSION:

Silver
nanoparticles were successfully synthesized by using green, cost-effective and
environmentally friendly manner. The reduction of silver ions by using Anacyclus
pyrethrum roots compound and stabilization of the AgNPs were thought to
occur due to the surface plasmon resonance with the ingredients present in the
plants roots extract. Formation of spherical shaped and well dispersed silver
nanoparticles with an average particle size of 19 to 34 nm was identified with
the help of TEM and XRD techniques. The FTIR data showed the biomolecules
present in synthesized nanoparticles were acting as capping as well as reducing
agents. The results explain that synthesized AgNPs showed antimicrobialactivity
against Salmonella typhi, Klebsiellapneumoniae, and Pseudomonas aeruginosaand
exhibit potential for medicines and other therapeutic applications.